New Research

Disorders of the Nervous System Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Inhibition by DARPP-321,2,3

Marina Polito,1,2 Elvire Guiot,1,2 Giuseppe Gangarossa,3,4,5 Sophie Longueville,2,6,7 Mohamed Doulazmi,1,2 Emmanuel Valjent,3,4 Denis Hervé,2,6,7 Jean-Antoine Girault,2,6,7 Danièle Paupardin-Tritsch,1,2 Liliana R. V. Castro,1,2 and Pierre Vincent1,2

DOI:http://dx.doi.org/10.1523/ENEURO.0060-15.2015 1CNRS, UMR8256 “Biological Adaptation and Ageing”, Institut de Biologie Paris-Seine (IBPS), F-75005 Paris, France, 2Université Pierre et Marie Curie (UPMC, Paris 6), Sorbonne Universités, Paris, F-75005, France, 3CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France, 4Institut National de la Santé et de la Recherche Médicale, U661, Montpellier, F-34094, France, 5Universités de Montpellier 1 & 2, UMR-5203, Montpellier, F-34094, France, 6Institut National de la Santé et de la Recherche Médicale UMR-S 839, Paris, France, and 7Institut du Fer a` Moulin, Paris, France

Abstract Type 10A (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium- sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP- dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for -1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs. Key words: biosensor imaging; cAMP; phosphodiesterase; protein kinase; schizophrenia; striatum

Significance Statement

The striatum is mainly composed of medium-sized spiny neurons that express either dopamine D1 receptors or dopamine D2 receptors. Their activity is associated with either the initiation of movement or action suppression, respectively. Biosensor imaging revealed that pharmacological inhibition of type 10A phosphodiesterase in-

creased cAMP levels in D1 and D2 neurons in the same manner, but only D2 neurons exhibited an increase in the protein kinase A-mediated phosphorylation level. This effect resulted from an asymmetrical regulation of

by DARPP-32. D2 neurons are thus more prone to respond to a tonic cAMP signal than D1 neurons, a property that may explain how phosphodiesterase 10A inhibitors produced antipsychotic-like

behavioral effects. This D1/D2 imbalance may also be critical for reward-mediated learning and action selection.

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Introduction Classical and atypical antipsychotic agents share the Schizophrenia is a devastating psychiatric disease, property of inhibiting D2 receptors and thus, in the stria- which results in persistent cognitive and emotional impair- tum, increase PKA-dependent phosphorylation selec- ments. Type 10A phosphodiesterase (PDE10A) inhibitors tively in D2 MSNs (Bateup et al., 2008; Bertran-Gonzalez were recently proposed as a treatment for schizophrenia et al., 2008, 2009). In contrast, psychostimulants, which (Kehler and Nielsen, 2011; Chappie et al., 2012); however, are psychotomimetic, activate many signaling responses their cellular mechanisms of action remain unclear with in D1 MSNs (Bateup et al., 2008; Bertran-Gonzalez et al., respect to their putative therapeutic effects. PDE10A is 2008, 2009). PDE10A inhibitors were shown to increase highly and almost exclusively expressed in medium-sized cAMP levels in the striatum (Schmidt et al., 2008) and spiny neurons (MSNs) of the striatum (Seeger et al., 2003; could be expected to mimic the effects of both antipsy- Coskran et al., 2006; Heiman et al., 2008; Lakics et al., chotic and psychotomimetic compounds. We used bio- 2010; Kelly et al., 2014). MSNs are divided into two cat- sensor imaging to precisely analyze the effects of PDE10A egories based on their expression of dopamine receptors inhibitors on cAMP/PKA signaling at the level of individual and their sites of projection, as follows: MSNs projecting D1 and D2 MSNs. Our work revealed that although PDE10A inhibition increased intracellular cAMP levels in to the substantia nigra highly express dopamine D1 re- both D and D MSNs, the downstream consequences at ceptors (hereafter termed D1 MSNs); whereas, MSNs pro- 1 2 jecting to the external globus pallidus highly express the level of PKA targets were profoundly different: the cAMP signal resulting from PDE10A inhibition strongly adenosine A2A and dopamine D2 receptors (hereafter increased PKA-dependent phosphorylation in D MSNs, termed D2 MSNs; Gerfen et al., 1990; Le Moine and Bloch, 2 1995; Bertran-Gonzalez et al., 2008; Matamales et al., whereas D1 MSNs remained mostly unaffected. Further 2009). The high expression of PDE10A in MSNs, and its analyses showed that the difference required DARPP-32- interaction with the scaffold protein AKAP150 (A-kinase dependent regulation of phosphatase activity in D1 and D2 anchoring protein 150), protein kinase A (PKA), PSD-95, MSNs. and NMDA receptor suggests an important role in mod- ulating the spread of the synaptic cAMP signals into the Materials and Methods cell body (Russwurm et al., 2015). Animals Besides PDE10A, striatal neurons express a number of Animals were housed under standardized conditions specific signaling proteins that markedly differ from those in with a 12 h light/dark cycle, stable temperature (22 Ϯ other brain regions (Girault, 2012), and that determine the 1ºC), controlled humidity (55 Ϯ 10%), and food and water characteristics of the cAMP/PKA signaling pathway (Castro available ad libitum. Homozygous mice expressing et al., 2013). Among these specific proteins, DARPP-32 DARPP-32 with the T34A or T75A mutation (Svenning- (32-kDa dopamine and cAMP-regulated phosphoprotein) is sson et al., 2003) were obtained by crossing heterozygous a multifunctional protein regulating phosphatase and kinase mice, on a mixed C57BL6/J-Sv129 background (a gift of activities: for example, when DARPP-32 is phosphorylated Dr. P. Greengard, The Rockefeller University, New York). at threonine 34 residue (Thr34) by PKA, it becomes a potent Male Drd2-EGFP heterozygous mice (C57Bl6/J) were inhibitor of serine/threonine protein phosphatase-1 (PP-1; generated as described previously (Gong et al., 2003). Hemmings et al., 1984; Svenningsson et al., 2004), increas- Experiments were performed in accordance with the reg- ing the duration of PKA-dependent signals (Castro et al., ulations under the control of the local ethic committee 2013). Charles Darwin C2EA - 05.

Live brain slice preparation Received June 1, 2015; accepted August 10, 2015; First published August 25, 2015. Brain slices were prepared from male mice that were 1The authors declare no competing financial interests. 8–12 days of age. Coronal brain slices were cut with a 2Author contributions: M.P., E.V., D.H., D.P.-T., L.R.V.C., and P.V. designed VT1200S microtome. Slices were prepared in an ice-cold research. M.P., E.G., G.G., S.L., and L.R.V.C. performed research. M.P., E.G., solution of the following composition (in mM): 125 NaCl, 0.4 G.G., S.L., M.D., L.R.V.C., and P.V. analyzed data. E.V., D.H., J.A.-G., D.P.-T., CaCl , 1 MgCl , 1.25 NaH PO , 26 NaHCO , 25 glucose, L.R.V.C., and P.V. wrote the paper. 2 2 2 4 3 3This work was supported by grants from ATIP-Avenir (Inserm) and from the and 1 kynurenic acid, saturated with 5% CO2 and 95% O2. Agence Nationale de la Recherche, ANR-2010-JCJC-1412) to EV and ANR09- The slices were incubated in this solution for 30 min and then MNPS-014 to DH, and ERC to JAG. The groups of PV, and JAG and DH are placed on a Millicell-CM membrane (Millipore) in culture part of the Bio-Psy Laboratory of Excellence. medium (50% Minimum Essential Medium, 50% HBSS, 6.5 Acknowledgments: Confocal microscopy and image analysis were per- formed at the Institut du Fer a` Moulin Imaging Facilities and at the Institute of g/L glucose, penicillin-streptomycin; Invitrogen). We used Biology Paris-Seine Imaging Facility (supported by the “Conseil Regional Ile-de the Sindbis virus as a vector to induce expression of the France”, the French National Research Council, and Sorbonne University, various biosensors after overnight incubation (Ehrengruber UPMC, Paris 6). et al., 1999). The coding sequences of Epac-SH150 (Polito Correspondence should be addressed to Pierre Vincent, UMR8256, 9, quai et al., 2013), AKAR2-NLS (Zhang et al., 2005), AKAR3 (Allen St. Bernard, F-75005 PARIS, France. E-mail: [email protected]. DOI:http://dx.doi.org/10.1523/ENEURO.0060-15.2015 and Zhang, 2006), and cygnet2 (Honda et al., 2001) were Copyright © 2015 Polito et al. inserted into the viral vector pSinRep5 (Invitrogen). The viral This is an open-access article distributed under the terms of the Creative vector (ϳ5 ϫ 105 particles per slice) was added, and slices Commons Attribution 4.0 International, which permits unrestricted use, distri- bution and reproduction in any medium provided that the original work is were incubated overnight at 35°C under an atmosphere properly attributed. containing 5% CO2. Before the experiment, slices were

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incubated for 30 min in the recording solution (125 mM NaCl, the ratio value coded in hue and the fluorescence of the

2mM CaCl2,1mM MgCl2, 1.25 mM NaH2PO4,26mM preparation coded in intensity. NaHCO3, and 25 mM glucose, saturated with 5% CO2 and Two-photon imaging was used to separate individual 95% O2). Recordings were performed with a continuous neurons for a precise quantification of the amplitude of perfusion of the same solution at 32°C. MSNs constitute the response (Figs. 1, 2). Ratio measurements were per- 95% of neurons in the striatum. Large neurons (smallest formed on a series of 5–10 consecutive image from the soma diameter, Ͼ14 ␮m), presumably cholinergic interneu- image stack, centered on the cell body. With cytosolic rons, were excluded. biosensors, when visible, the nucleus was excluded from the measurement. Wide-field imaging (Figs. 3A–E) also Live brain slice imaging allowed the unambiguous identification of D1 and D2 For two-photon imaging, excitation was obtained using MSNs, provided that the infection level was kept low and a Ti:sapphire laser (MaiTai HP; Spectra Physics) tuned at no fluorescence overlap between neighboring neurons 850 nm for CFP excitation. Galvanometric scanners was observed. The optical cross-contamination resulting (model 6210; Cambridge Technology) were used for ras- from out-of-focus light was evaluated by the final re- ter scanning, and a piezo-driven objective scanner (P-721 sponse to CGS 21680 and SKF-38393, applied sequen- PIFOC; Physik Instrumente GmbH) was used for z-stack tially: cells were rejected from analysis if the cross- image acquisition. The system was controlled by MP- contamination was Ͼ30%. For cGMP imaging (Fig. 3F,G), scope software (Nguyen et al., 2006). The microscope the data were quantified as relative ratio change. was based on an Olympus BX51WI upright microscope with a 40ϫ 0.8 numerical aperture (NA) or 60ϫ 0.9 NA Quantifications of cAMP signals water-immersion objective. A two-photon emission filter The amplitudes of responses were quantified for each was used to reject residual excitation light (E700 SP; neuron as the fractional change in ratio from its own Chroma Technology). A fluorescence cube containing baseline and maximal final ratio response. Responses 479/40 and 542/50 emission filters and a 506 nm dichroic obtained from MSNs of the same type were averaged for beamsplitter (FF01-479/40, FF01-542/50 and FF506- each experiment (i.e., brain slice). Di02-25x36 Brightline Filters; Semrock) was used for The free cAMP concentrations were estimated with the the orthogonal separation of the two fluorescence sig- Epac-SH150 biosensor from the ratio measurement using ␮ nals. Two imaging channels (H9305 photomultipliers; the Hill equation, with a Kd of 4.4 M and a Hill coefficient Hamamatsu) were used for simultaneous detection of the of 0.77, as determined from Polito et al. (2013). The two types of fluorescence emission. For each data point, an maximal response corresponding to biosensor saturation ␮ image stack of 30–40 images with a 0.5 m interval was (Rmax) was determined for each neuron at the end of the acquired. recording. This level was obtained by applying 13 ␮M Wide-field images were obtained with an Olympus forskolin (FSK), a dose known to be sufficient to maximally BX50WI or BX51WI upright microscope with a 40ϫ 0.8 NA phosphorylate the highly sensitive probe AKAR3 in MSNs.

water-immersion objective and an ORCA-AG Camera For cAMP biosensors, this Rmax value was obtained with (Hamamatsu). Images were acquired with iVision (Biovi- 200 ␮M IBMX and 13 ␮M forskolin. sion). The excitation and dichroic filters were D436/20 and The baseline cAMP level in control conditions was eval- 455dcxt. Signals were acquired by alternating the emis- uated by inhibiting adenylyl cyclases with 200 ␮M sion filters, HQ480/40 for CFP, and D535/40 for yellow SQ22536, which resulted in a ratio decrease, measured in fluorescent protein, with a filter wheel (Sutter Instru- wide-field microscopy, of Ϫ4.0% of the maximal ratio ments). These filters were obtained from Chroma Tech- change. This decrease in baseline ratio was Ϫ4.9 Ϯ 0.7 nology. and Ϫ3.3 Ϯ 0.7 (n ϭ 6, p Ͻ 0.05 with paired Student’s t

No correction for bleed-through or direct excitation of test), respectively, in D1 and D2 MSNs. Assuming that the acceptor was applied, since this correction, while adenylyl cyclase inhibition effectively decreased cAMP increasing the absolute amplitude of ratio changes, also levels down to a level sufficient to reach the minimal ratio

increases the noise in the measurement (Ducros et al., level (Rmin), these values suggest a baseline cAMP con- 2009). centration in a range of ϳ100 nM. The biosensor chromophores are sensitive to nonspe- cific environmental disturbances. We used a mutated ver- Tissue preparation and immunofluorescence sion of AKAR3 in which the threonine residue of the PKA Mice, 8-10 weeks old, were treated with the drug for 60 phosphorylation site was replaced with an alanine residue min and then rapidly anesthetized with pentobarbital (500 (T391A) as a control. This AKAR3 (T391A) control sensor mg/kg, i.p.; Sanofi-Aventis) and were transcardially per- reported no ratio change in response to PDE10A inhibition fused with 4% (w/v) paraformaldehyde in 0.1 M PBS, pH in MSNs. 7.5. Brains were post-fixed overnight in the same solution and stored at 4°C. The 30-␮m-thick sections were cut Data analysis with a vibratome and stored at Ϫ20°C in a solution con- Images were analyzed with custom routines written in taining 30% (v/v) ethylene glycol, 30% (v/v) glycerol and the IGOR Pro environment (Wavemetrics). The emission 0.1 M sodium phosphate buffer, until they were processed ratio was calculated for each pixel, as follows: F535/F480 for immunofluorescence. Sections were processed as de- for AKAR2-NLS and AKAR3, and F480/F535 for cygnet2 scribed in Bertran-Gonzalez et al. (2009). Sodium fluoride H150 and Epac-S sensors. The pseudocolor images display 0.1 mM was included in all buffers and incubation solu-

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A C

B D

Figure 1. PDE10A inhibition increases cAMP levels in both in D1 and D2 MSNs, and PKA-dependent phosphorylation only in D2 MSNs. A, MSNs in a neostriatal mouse brain slice expressing the cAMP biosensor Epac-SH150 were imaged with two-photon microscopy during the application of PQ-10 (100 nM). Images (vertical projection of the image stack) show the raw fluorescence at 535 nm (left, in grayscale) and the ratio (in pseudocolor) indicating intracellular cAMP concentrations, at the times indicated by the arrows on the graph below. The calibration square in A indicates the spatial scale (the size of the square is indicated in micrometers), and shows the ranges of intensity (horizontally) and ratio (vertically). Each trace on the graph indicates the F480/F535 emission ratio measured in regions indicated by the color contour drawn on the raw image. Traces in gray correspond to regions that are not visible on these

images. Traces are plotted in two groups according to their response to either CGS 21680, an adenosine A2A receptor agonist (CGS, ␮ ␮ 1 M), or SKF-38393, a D1-like receptor agonist (SKF, 1 M). The thick black line represents the average of all the traces in a group. FSK (13 ␮M) and IBMX (200 ␮M) were applied at the end of the recording to determine the maximal response. B, The same experiment

was repeated for every PQ-10 concentration tested. No significant difference was found between D1 and D2 MSNs (two-way ANOVA: ϭ Ͻ ؊4 ϭ ϭ ϫ ϭ ϭ dose effect, F(6,54) 40.91, p 10 ;D1/D2 effect, F(1,54) 2.56, p 0.115; dose D1/D2 interaction, F(6,54) 0.625, p 0.709). Error bars indicate the SEM. C, Same as A, except that the AKAR3 biosensor was used to monitor PKA-dependent phosphorylation, and the ratio was calculated as F535/F480. D, Same as B for AKAR3 measurements. Data were analyzed with two-way ANOVA: dose ϭ Ͻ ؊4 ϭ Ͻ ؊4 ϫ ϭ Ͻ ؊4 effect, F(6,38) 28.31, p 10 ;D1/D2 effect, F(1,38) 143.73, p 10 ; dose D1/D2 interaction, F(6,38) 9.23, p 10 . p Ͻ 0.001ءءء ,Bonferroni’s post hoc test

tions. Histone H3 phosphorylation was revealed with a catalog #A10262; Life Technologies). Following incuba- rabbit polyclonal antibody against phospho-Ser10-H3 (1: tion with primary antibodies, sections were rinsed three 1000; catalog #06570; Millipore) the specificity of which times for 10 min in TBS and incubated for 45–60 min with was confirmed in a previous study (Jordi et al., 2013). GFP goat Cy3-coupled (1:500; Jackson ImmunoResearch; Fig. was detected using chicken antibody against GFP (1:500; 5) and goat A488 (1:500; Life Technologies; Fig. 6). Sec-

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tions were rinsed for 10 min twice in Tris-buffered saline PDE10A inhibition with PQ-10 (100 nM) increased cAMP and twice in Tris buffer (0.25 M Tris) before mounting in levels in all MSNs (Fig. 1A) in a dose-dependent manner. 1,4-diazabicyclo-[2. 2. 2]-octane (Sigma-Aldrich). This dose dependency was not statistically different be-

Single-labeled images (Fig. 6) were obtained with a tween D1 and D2 MSNs (Fig. 1B). At the highest doses (1 Zeiss LSM780 Confocal Microscope. Double-labeled im- and 2 ␮M), cAMP responses reached ϳ35% of the max- ages (Fig. 5) were obtained with a Leica TCS SPE Con- imal response to FSK plus IBMX, which corresponds to a focal Microscope with laser lines at 496 and 561 nm, concentration of free cAMP of ϳ2 ␮M (see Materials and acquiring in the 501–539 nm and 570–660 nm bands. All Methods for details on the estimation of cAMP concen- parameters were held constant for all sections from the trations). These experiments showed that, in the basal same experiment. condition, cAMP is tonically produced in striatal slices and that PDE10A contributes significantly to its degradation. Statistics Data were analyzed with SPSS statistical software version PDE10A inhibition increases the phosphorylation of

22.0. Normality in variable distributions and homogeneity a PKA target exclusively in D2 MSNs of variances across groups were assessed with the Sha- We then analyzed the effects of PQ-10 on PKA- piro–Wilk and Levene tests, respectively. Variables that dependent phosphorylation levels using the PKA biosen- failed any of these tests were analyzed with nonparamet- sor AKAR3. As for cAMP imaging, MSNs were identified ric statistics using the Kruskal–Wallis ANOVA on ranks at the end of each experiment by their response to either

followed by Mann–Whitney rank sum test with a Dunn– A2A or D1 receptor agonist. The maximal AKAR3 response Sidak adjustment test for pairwise multiple comparisons. was elicited by FSK. Although the increase in free cAMP

Variables that passed the normality test were analyzed concentration was similar in D1 and D2 MSNs (Fig. 1A,B), with ANOVA followed by Bonferroni post hoc test for the resulting PKA-dependent phosphorylation levels were multiple comparisons or by Student’s t test for comparing completely different (Fig. 1C): PQ-10 (100 nM) strongly

two groups. Paired data were analyzed with a Student’s t increased the emission ratio of AKAR3 in the D2 MSNs (66 test. A p value of Ͻ0.05 was used as a cutoff for statistical Ϯ 4% of the maximal response to FSK, n ϭ 4); whereas, significance. All error bars represent the SEM; n indicates Ϯ in D1 MSNs, the ratio remained at a much lower level (6 the number of experiments (i.e., the number of brain slices 2%). These results indicated a significantly higher phos- tested), with at least four neurons of each type in each phorylation of AKAR3 in D2 than in D1 MSNs in response experiment. All experiments were performed on at least to PQ-10. The effect of PQ-10 on AKAR3 ratio in D2 MSNs three different brain slices from at least two animals. was steeply dose dependent with a maximal effect Ͻ reached at 100 nM (Fig. 1D). In stark contrast to D2 Drugs MSNs, even high doses of PQ-10, which increased cAMP

SKF-38393 hydrobromide, CGS 21680 hydrochloride, to the same levels in D1 and D2 MSNs (Fig. 1B), only 1-methyl-3-isobutylxanthine (IBMX), rolipram, papaver- produced a very small effect on AKAR3 phosphorylation ine, roscovitine, okadaic acid, cantharidin, gabazine, in D1 MSNs (Fig. 1D). These experiments thus revealed a CNQX, APV, and forskolin were obtained from Tocris much stronger effect of cAMP on PKA-dependent phos- Cookson. TTX was from Latoxan. PQ-10, MP-10, and phorylation in D2 than in D1 MSNs. roflumilast were a gift from Janssen Pharmaceuticals. PDE10A may be addressed differentially in the cyto- TP-10 was provided by Pfizer through the Compound plasm and membranes (Kotera et al., 2004; Charych et al., Transfer Program. 2010), and cAMP dynamics could differ in subcellular domains of different geometry, like dendrites (Castro Results et al., 2010). An increase in AKAR3 ratio was observed PDE10A inhibition reveals a tonic cAMP production exclusively in the dendritic branches that responded to

in both D1 and D2 MSNs the A2A agonist, whereas dendrites, which responded to M Since both D1 and D2 MSNs express high levels of the D1 agonist, showed no response to 100 n PQ-10 PDE10A protein (Nishi et al., 2008), we used biosensor- (Fig. 2A). This is consistent with other biosensor record- imaging approaches to compare the effects of PDE10A ings in which dendrites of D1 MSNs also exhibited no

inhibition on the cAMP/PKA signaling cascade in D1 and baseline response to PDE10A inhibition (Yagishita et al., D2 MSNs. First, we monitored changes in intracellular 2014). cAMP concentrations with two-photon microscopy in stri- Once activated, PKA can translocate to the nucleus and atal brain slices expressing Epac-SH150. At the end of phosphorylate a number of nuclear proteins. We exam-

every experiment, an agonist of adenosine A2A receptors ined whether the differential response to PQ-10 also ex- ␮ (CGS 21680, 1 M) and an agonist of dopamine D1 recep- isted in the nucleus. Using the nuclear AKAR2-NLS tors (SKF-38393, 1 ␮M) were applied sequentially, trigger- biosensor, we found that PDE10A inhibition induced a

ing a positive cAMP response in D2 and D1 MSNs, strong ratio increase in D2 MSNs, while D1 MSNs re- respectively, thereby functionally identifying MSN sub- mained unresponsive (Fig. 2B). These results showed that types. The final application of the general adenylyl cyclase PQ-10 efficiently increased AKAR3 phosphorylation in the

activator FSK (13 ␮M) together with the nonselective cytoplasm and nucleus of D2 but not D1 MSNs, whereas phosphodiesterase inhibitor IBMX (200 ␮M) produced the they had a similar and selective effect on cAMP produc- maximal ratio response used for normalization. tion in the two cell types.

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A B

Figure 2. PDE10A inhibition triggers positive PKA responses in dendrites and nuclei preferentially in D2 MSNs. A, B, Brain slices expressed the PKA sensor AKAR3 (A) or AKAR2-NLS (B) and were imaged by two-photon microscopy during the application of PQ-10 (100 nM). Images show the raw fluorescence at 535 nm (left in grayscale) and the ratio (in pseudocolor) indicating the PKA-dependent phosphorylation level of the biosensor, at the times indicated by the arrows on the graph below. The calibration square in A indicates the spatial scale (above, in micrometers), and shows the ranges of intensity (horizontally) and ratio (vertically). Each trace on the graph indicates the F535/F480 emission ratio measured on regions indicated by the color contour drawn on the raw image. Traces are plotted in two groups according to their response to either CGS 21680 (CGS, 1 ␮M) or SKF-38393 (SKF, 1 ␮M). The thick black line represents the average of all the traces in a group. FSK (13 ␮M) was applied at the end of the recording to determine the maximal response.

PDE10A inhibition effects are antagonized by D2 that the positive AKAR3 response to PQ-10 was specific receptors and independent of A2A receptors to MSNs expressing D2 receptors. One feature of striatopallidal MSNs is the coexpression of We then determined whether the effect selectivity for D2 D2 dopamine and A2A adenosine receptors, negatively MSNs was a particular property of PQ-10 or was also and positively coupled to adenylyl cyclase, respectively observed with other PDE10A inhibitors. The effects of (Schiffmann et al., 1991; Schiffmann and Vanderhaeghen, MP-10 (100 nM) and (1 ␮M) on AKAR3 ratio 1993; Ferré et al., 1997; Svenningsson et al., 1999; Ba- were similar (Fig. 3C,D), inducing an AKAR3 ratio increase

teup et al., 2008; Bertran-Gonzalez et al., 2009). Applica- selectively in D2 MSNs. TP-10 also produced the same ␮ tion of the D2 receptor agonist quinpirole (1 M) response profile (see below). In contrast, PDE4 inhibitors completely reversed the PQ-10-induced AKAR3 response (rolipram, 100 nM, n ϭ 4; and roflumilast, 1 ␮M, n ϭ 4) had

in D2 MSNs, monitored with wide-field microscopy (Fig. no effect on basal AKAR3 ratio (data not shown). 3A). Application of quinpirole alone had no effect on the As D2 MSNs express adenosine A2A receptors, we ex- basal AKAR3 ratio but prevented positive responses to amined whether the tonic presence of extracellular aden- Ϯ ϭ PQ-10 in D2 MSNs with 11 3% (n 5) of the maximal osine in our brain slice preparation activated adenylyl Ϯ FSK response in D2 MSNs compared with 9 2% in D1 cyclase and might be responsible for the positive re- MSNs (Fig. 3B). These results showed that the activation sponse to PQ-10 recorded specifically in these MSNs.

of D2 receptors opposed the effect of PQ-10 in D2 MSNs, When A2A receptors were blocked with SCH 58261 (100 most likely via Gi-mediated inhibition of the tonic adenylyl nM), PQ-10 still elicited positive responses in D2 MSNs cyclase activity. These experiments also further confirmed (Fig. 3E). In contrast, SCH 58261 blocked the responses

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A B

CDE

F G

Figure 3. A, Activation of D2 dopamine receptors suppressed the effect of PDE10A inhibition on AKAR3 ratio. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 ␮M) or CGS 21680 (CGS, 1 ␮M), respectively. The thick black line represents the average of all the traces in a ␮ group. Bath application of the agonist of dopamine D2 receptors quinpirole (1 M) reversed the response to PQ-10 (100 nM). B,D2 receptor activation prevented the response to PDE10A inhibition: the effect of PQ-10 was measured in the presence of the D2 agonist ␮ Ͼ ϭ quinpirole (1 M). No statistically significant difference (p 0.05) was found between D1 and D2 MSNs (n 5). The effect of PQ-10 is displayed for comparison on the left (same data as in Fig. 4E). C, D, Other PDE10A inhibitors also increased the AKAR3 ratio ϭ ␮ ϭ preferentially in D2 MSNs: MP-10 (C; 100 nM, n 9) and papaverine (D;1 M, n 5) both increased the AKAR3 ratio selectively in ␮ D2 MSNs. E, PQ-10 increased AKAR3 ratio selectively in D2 MSNS even when adenosine A2A receptors were inhibited with 100 M p Ͻ 0.001. F, PDE10A inhibition had noءءء .SCH 58261 (n ϭ 4). B–E, Statistical differences were tested with paired Student’s t test effect on cGMP levels measured with the cGMP sensor cygnet2. The NO donor DEANO (100 ␮M) increased the ratio; after reaching a steady-state level, PQ-10 (1 ␮M) was added; at the end of the recording, the maximal ratio response was elicited by DEANO plus

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continued IBMX (200 ␮M). G, No difference was measured when comparing the response with DEANO alone and DEANO with PQ-10, while IBMX Ϯ produced a significant increase. The data expressed as the mean SEM were analyzed by repeated-measures one-way ANOVA F(1,5) p Ͻ 0.01). A–G, Brain slices were imaged with wide-fieldءء :ϭ 11,224, p Ͻ 0.001, n ϭ 6, followed by Bonferroni’s post hoc test microscopy. All data are expressed as the mean Ϯ SEM.

to the A2A agonist CGS 21680 (data not shown). These pooled (Fig. 4F, gray color bar). To identify which phos- results showed that tonic activation of A2A receptors was phatase subtype was involved in the response, we used not required for the phosphorylation response to PQ-10 in fostriecin (200 nM), a selective inhibitor of PP-2A (Swingle

D2 MSNs. We then examined whether the phosphoryla- et al., 2009). We observed no effect of this inhibitor alone tion signal involved endogenous neuronal activity, or re- on basal AKAR3 ratio, and it did not alter the selective

quired the glutamate and GABA which may be present in response to PQ-10 in D2 MSNs (Fig. 4B). These results the brain slice. This was not the case since, in the pres- showed that PP-2A was not involved in the different

ence of blockers of voltage-gated sodium channels (TTX, responsiveness of D1/D2 and rather suggested the in- ␮ 100 nM), calcium channels (CdCl2, 200 M), non-NMDA volvement of PP-1. receptors (CNQX, 10 ␮M), NMDA receptors (APV, 10 ␮M), ␮ and GABAA receptors (SR 95531, 1 M), the selective PP-1 regulation by DARPP-32 is necessary for the Ϯ effect of PQ-10 on D2 MSNs was still present (25 3vs selective responsiveness of D2 MSNs to PDE10A Ϯ 71 10 of the maximal FSK response in D1 and D2 MSNs, inhibition respectively; n ϭ 3, paired Student’s t test; data not DARPP-32 is expressed at high levels in both types of shown). MSNs and constitutes a powerful and specific inhibitor of Since PDE10A also degrades cGMP and PDE10A inhi- PP-1 when it is phosphorylated at Thr34 (Hemmings et al., bition was shown to increase cGMP levels and affect 1984). We used a knock-in mutant mouse line in which synaptic transmission in vivo (Siuciak et al., 2006; Thr34 is replaced by an alanine (T34A; Svenningsson Schmidt et al., 2008; Grauer et al., 2009; Padovan-Neto et al., 2003). In T34A mice, the effect of PQ-10 on AKAR3 Ϯ ϭ et al., 2015), we used wide-field imaging of the cGMP was strongly reduced in D2 MSNs (14 2%, n 13, 6 biosensor cygnet2 (Honda et al., 2001) to determine mice), whereas, as in wild-type mice, no effect was ob- Ϯ whether PDE10A also regulated cGMP in MSNs. PQ-10 (1 served in D1 MSNs (8 1%; Fig. 4C,E). Normal responses ␮ ϭ ϭ M) had no effect on the baseline cGMP levels (n 4, p to D1 or A2A stimulations were observed at the end of the 0.44, one-sample Student’s t test). In addition, PQ-10 (1 recording. The phosphatase inhibitor cantharidin un- ␮M) had no effect on the cGMP steady-state level ob- masked the response to PQ-10 in all MSNs of the tained with the nitric oxide (NO) donor DEANO (Diethyl- DARPP-32 T34A mice (Fig. 4F), confirming that, upon amine nitric oxide, 100 ␮M; n ϭ 6; Fig. 3F), while the inhibition of PP-1, PQ-10 was still capable of increasing nonspecific phosphodiesterase inhibitor IBMX produced the AKAR3 response in these mutant mice. Together, a significant ratio increase. These results indicated that in these results show that the inhibition of PP-1 by our conditions PDE10A inhibition does not significantly DARPP-32 is necessary for the difference in responsive-

affect cGMP levels in MSNs. ness of D1 and D2 MSNs. DARPP-32 also inhibits PKA activity when it is phos-

Different responsiveness of D1 and D2 MSNs is phorylated at Thr75 by Cdk5 (Bibb et al., 1999; Nishi et al., abolished by protein phosphatase inhibition 2000). A higher phosphorylation level of this residue in D1 The difference between D1 and D2 MSNs in the phosphor- MSNs could be responsible for a weaker PKA activity in ylation level of AKAR3 could result from differences in the these neurons. However, in a knock-in mutant mouse line rate of phosphorylation by PKA, dephosphorylation by with a Thr75-to-alanine mutation (DARPP-32 T75A; Sven- phosphatases, or both. A difference in PKA levels is un- ningsson et al., 2003), the profile of the AKAR3 response likely because immunostaining of catalytic subunits in the to PQ-10 was the same as that in wild-type mice (Fig. striatum did not reveal major differences between cells 4D,E). Moreover, the Cdk5 inhibitor roscovitine (10 ␮M)

(Yang et al., 2014). Since AKAR3 biosensor responses to had no effect on the D1/D2 imbalance in the response to PKA activation are reversed by the action of endogenous PQ-10 (Fig. 4G), ruling out the involvement of Thr75 of protein phosphatases (Gervasi et al., 2007), we investi- DARPP-32 as a critical determinant for the lack of PQ-10-

gated the role of protein phosphatases in the different dependent AKAR3 responses in D1 MSNs. responsiveness of D1 and D2 MSNs to PDE10A inhibitors. Cantharidin (30 ␮M), a nonselective inhibitor of PP-1 In vivo PDE10A inhibition selectively induces histone

and PP-2A, had no effect by itself on the AKAR3 emission H3 phosphorylation in D2 MSNs of the dorsomedial ratio (Fig. 4A). However, when PQ-10 (100 nM) was ap- striatum plied in the bath (Fig. 4A), the AKAR3 ratio increased in Our results showed a marked difference in the respon- Ϯ virtually all D1 and D2 MSNs (79 10% of the FSK siveness of D1 and D2 MSNs to PDE10A inhibitor in slices. ϭ response, n 5). Since these responses did not return to We then investigated whether the D1/D2 imbalance could the baseline after drug washout, it was impossible to also be observed in vivo by monitoring phospho-histone

distinguish between D1 and D2 MSNs, and all MSNs were H3 at Ser10 residue (PH3), a substrate for several protein

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A B

C D

E F G

Figure 4. DARPP-32-mediated phosphatase inhibition favors PKA signaling in D2 MSNs. A, PP-1 and PP-2A were inhibited with cantharidin. Cantharidin (30 ␮M) alone did not change the basal ratio but strongly increased the AKAR3 response to PQ-10 (100 nM)

in all MSNs. These responses were not reversible, making the final identification of D1 and D2 MSNs impossible (gray bars in F, which represent the responses of all MSNs). B,D2 MSNs responded selectively to PQ-10 (100 nM) even when the PP-2A inhibitor fostriecin (p Ͻ 0.01). C–E, Mutation of the Thr34 to Ala in DARPP-32 (DARPP-32 T34Aءء ;nM) was applied (n ϭ 4, paired Student’s t test 200)

strongly reduced the effect of PQ-10 (100 nM)inD2 MSNs, whereas the selective effect of PQ-10 on D2 MSNs remained in brain slices from animals bearing the Thr75 to Ala mutation in DARPP-32 (DARPP-32 T75A). C, D, Representative experiments performed with DARPP-32 T34A (C) and DARPP-32 T75A (D) knock-in mice. Each trace on the graph indicates the ratio measurement on MSNs ␮ expressing AKAR3 and is identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 M) or CGS 21680 (CGS, 1 ␮M), respectively. The thick black line represents the average of all the traces in each group. E, The data expressed as the mean Ϯ ϭ Ͻ ؊4 ϭ Ͻ ؊4 SEM were analyzed by two-way ANOVA: genotype effect, F(2,72) 71.12, p 10 ;D1/D2 effect, F(1,72) 333.07, p 10 ; Ͻ ءءء ؋ ϭ Ͻ ؊4 genotype D1/D2 interaction, F(2, 72) 49.53, p 10 . Bonferroni’s post hoc test: p 0.001. F, In wild-type (WT) mice and DARPP-32 T34A mutants, and in the presence of cantharidin (30 ␮M), all MSNs responded to PQ-10 (100 nM) with an increase in ϭ AKAR3 ratio such that D1 and D2 MSNs could not be distinguished (n 5 for both). No significant difference was obtained between

July/August 2015, 2(4) e0060-15.2015 eNeuro.sfn.org New Research 10 of 15 continued Ͼ wild-type and DARPP-32 T34A mutant (unpaired Student’s t test, p 0.05). G,D2 MSNs responded selectively to PQ-10 (100 nM) .(p Ͻ 0.05ء ;even when the Cdk5 inhibitor roscovitine (10 ␮M) was applied (n ϭ 4, paired Student’s t test

kinases including PKA (Nowak and Corces, 2004). We phorylation response in D2 MSNs than in D1 MSNs. We used transgenic mice in which D2 MSNs are identified by provide evidence that this difference is present in all GFP fluorescence (drd2-EGFP mice; Gong et al., 2003; striatal regions in brain slices and in the dorsomedial Bertran-Gonzalez et al., 2008) and monitored PH3 by striatum of adult mice in vivo. Moreover, we show that the immunofluorescence. In these in vivo experiments, we regulation of PP-1 activity by DARPP-32 is required for studied the effects of TP-10 (3 mg/kg, i.p.), a PDE10A these specific effects of PDE10A inhibitors. These obser- inhibitor known to produce clear behavioral effects vations provide novel insights into the regulation of the (Schmidt et al., 2008). At this dose, TP-10 induced a large cAMP/PKA pathway in the two populations of MSNs and increase in the number of PH3-positive neurons in the the possible antipsychotic action of PDE10A inhibitors. striatum 60 min after treatment, compared with vehicle PDE10A is one of the specifically enriched in treatment. Quantification showed that 93% of the PH3- the striatum (Seeger et al., 2003; Coskran et al., 2006; immunoreactive neurons were GFP positive (D2 MSNs) in Heiman et al., 2008; Lakics et al., 2010; Kelly et al., 2014), the dorsomedial striatum, whereas in lateral parts of the and the data reported here show that PDE10A plays an striatum both GFP-negative and GFP-positive D2 MSNs important role in degrading basally produced cAMP in exhibited PH3 immunoreactivity (Fig. 5A–C). In the nu- both D1 and D2 MSNs. MSNs thus contrast with pyramidal cleus accumbens, a sparse and irregular labeling was neurons of the prefrontal cortex in which basal cAMP is observed in the shell region, and no immunoreactivity was predominantly controlled by PDE4 (Castro et al., 2010). detected in the core. In all striatal regions, no PH3 immu- Although PDE4B is also expressed in D2 MSNs (Nishi noreactivity was observed in large neurons expressing et al., 2008), biosensor imaging of the somatic cytoplasm low levels of GFP, presumably corresponding to cholin- did not reveal significant effects of its inhibition in MSNs in ergic interneurons. our conditions. Since in vivo the selective effects of TP-10 on D2 MSNs PDE10A inhibition showed no effect on basal or stimu- was observed in the dorsomedial, but not the dorsolateral lated cGMP production. A similar lack of effect of PDE10A striatum, we performed a set of experiments in brain inhibitors on cGMP has already been reported in brain slices to compare medial and lateral dorsal striatum using slice preparations (Nishi et al., 2008). PDE10A thus differs the same inhibitor. The AKAR3 responses to TP-10 (100 from PDE1 and PDE2, other dual-specificity PDEs ex- nM) were similar, with a strong effect of TP-10 in D2 MSNs pressed in MSNs, which were shown in striatal homoge- but not in D1 MSNs (Fig. 5D,E). Altogether, these experi- nates to be the major PDEs involved in the control of ments showed that PDE10A exerts selective effects on D 2 cGMP levels (Russwurm et al., 2015). Using biosensor- MSNs in striatal slices and that this selectivity is main- imaging techniques, PDE2 was also shown to be the main tained in vivo in the dorsomedial striatum. PDE that regulated stimulated cGMP, while also regulat- ing cAMP in a cGMP-dependent manner (Polito et al., DARPP-32 is required for the in vivo effects of TP-10 2013). In vivo, PDE10A inhibition was shown to increase Our experiments in striatal slices showed that the inhibi- cGMP levels and to affect synaptic transmission (Siuciak tion of PP-1 by DARPP-32 phosphorylated on Thr34 was et al., 2006; Schmidt et al., 2008; Grauer et al., 2009; necessary for the selective responsiveness to PDE10A Padovan-Neto et al., 2015), and why this effect was not inhibition on AKAR phosphorylation. We then investigated observed in brain slices remains to be determined. One whether the effects of PDE10A inhibition in vivo also depended on the phosphorylation of DARPP-32 at Thr34. hypothesis to explain this discrepancy might be that, in T34A knock-in and wild-type littermates were treated with vivo, PDE10A inhibitors recruit nitric oxide synthase- TP-10 (3 mg/kg, i.p.) or vehicle (four animals for each positive striatal interneurons through a global increase in condition) and brain sections analyzed by immunofluores- network activity. cence for PH3 60 min after injection. Whereas wild-type While D1 and D2 MSNs share a number of cellular littermates strongly responded to TP-10, the effect of features, more detailed studies revealed subtle differ- TP-10 was completely abolished in the DARPP-32 T34A ences (Valjent et al., 2009), such as different excitability mutant mice in all regions of the striatum (Fig. 6A,B). profiles (Gertler et al., 2008; Threlfell et al., 2009). Differ- These experiments clearly showed that Thr34 in ences were also reported at the level of PKA-dependent DARPP-32 was required for the effects of PDE10A inhib- phosphorylation of GABAA receptors and DARPP-32, which were higher in D MSNs than in D MSNs (Janssen itor on D1 and D2 MSNs in both the dorsomedial and 2 1 dorsolateral striatum in vivo. et al., 2009; Nishi et al., 2008). Our work reveals a possible basis for these D1/D2 differences, which lies at the level of Discussion DARPP-32-mediated PP-1 regulation. In D1 MSNs, the Our study shows that although PDE10A is expressed and Thr34 of DARPP-32 is in a lower phosphorylation state functional in all types of MSNs, its inhibition in striatal than in D2 MSNs and, thus, the PP-1 activity reverts PKA slices produces a higher PKA-dependent protein phos- target sites to the dephophorylated state (Fig. 7). This

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AB

C P

D E

Figure 5. In vivo effects of PDE10A inhibition by TP-10. A, In the medial part of the dorsal striatum of drd2-EGFP adult mice treated

with TP-10 (3 mg/kg), PH3 was selectively observed in D2 MSNs. In the lateral part of the dorsal striatum, PH3 immunoreactivity was observed in both EGFP-positive and EGFP-negative MSNs. EGFP and PH3 are shown in grayscale, and are overlaid with EGFP in ␮ ϩ green and PH3 in red (Merge). Scale bar, 20 m. B, Each color spot represents a position where the relative distribution of D2/(D1 D2) PH3-positive MSNs is indicated in pseudocolor, over a schematic of coronal mouse brain (Franklin and Paxinos, 2007). C, PH3-positive nuclei were quantified in medial and lateral parts of the dorsal striatum as defined by the dotted line in B. The effect of localization was significant (Kruskal–Wallis test followed by a Mann–Whitney test with a Dunn–Sidak adjustment test for pairwise ءءء Ͻ Ϫ4 multiple comparisons tests, p 10 ), with PH3-positive nuclei being preferentially D2 MSNs in the medial striatum. indicates a Ͻ ؊4 difference between EGFP-positive (D2) and EGFP-negative (D1) MSNs with p 10 . D, The preferential AKAR3 response is also observed in the lateral striatum in brain slices from neonate mice. MSNs were transduced for the expression of the AKAR3 biosensor and imaged with wide-field microscope in the lateral striatum. Each trace on the graph indicates the ratio measurement on MSNs ␮ expressing AKAR3 and was identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 M) or CGS 21680 (CGS,

July/August 2015, 2(4) e0060-15.2015 eNeuro.sfn.org New Research 12 of 15 continued 1 ␮M), respectively. The thick black line represents the average of all the traces in each group. TP-10 (100 nM) increased AKAR3 ratio selectively in D2 MSNs. E, The same experiment was repeated: there was no effect of localization, and TP-10 increased the AKAR3 ϭ ratio selectively in D2 MSNs in both the dorsolateral and dorsomedial striatum (two-way ANOVA: localization effect, F(1,12) 0.374, ϭ ϭ Ͻ ؊4 ϫ ϭ ϭ p 0.374; D1/D2 effect, F(1,12) 44.01, p 10 ; localization D1/D2 interaction, F(1,12) 0.042, p 0.804. Bonferroni’s post hoc .p Ͻ 0.01.). C, E, Error bars indicate the SEMءء :test

hypothesis implies that a powerful mechanism prevents such as that produced by PDE10A inhibition, activates DARPP-32 from remaining phosphorylated on the Thr34 PKA, and, because PP-1 is inhibited, PKA targets remain position selectively in D1 MSNs. Thr34 residue is effi- phosphorylated. Indeed, when DARPP-32 bears the T34A ciently dephosphorylated by both PP-2A and PP-2B (but mutation and can no longer inhibit PP-1, D2 MSNs fail to not by PP-1; Nishi et al., 1999), and further work is needed respond to PDE10A inhibition (Fig. 7). to analyze the possible differences in PP2A and PP2B Further work is needed to analyze the possible differ- activities between D1 and D2 MSNs. In contrast to tonic ences in PP-2A and PP-2B activities between D1 and D2 cAMP levels induced by PDE10A inhibition, cAMP signals MSNs that may contribute to the higher level of elicited by D1 receptor stimulation lead to a phosphoryla- DARPP-32 phosphorylation on Thr34. tion of T34 and inhibition of PP-1 (Bateup et al., 2008), an Another potential player in the D1/D2 differences is the effect that is also clearly visible on transient responses to phosphorylation of DARPP-32 on Thr75, which is cata- dopamine stimulations (Castro et al., 2013). This is con- lyzed by Cdk5 and is responsible for PKA inhibition (Bibb sistent with the observation that, in D1 MSNs, PDE10A et al., 1999). Since Thr75 is dephosphorylated by a PKA- inhibition only affects PKA-dependent modulation of syn- activated form of PP-2A containing the B56 subunit (Ahn aptic transmission when cAMP production is stimulated et al., 2007), it could contribute to a hypersensitive feed-

(Mango et al., 2014). This nonlinearity in D1 MSNs may forward loop. However, this mechanism did not appear to improve the detection of powerful but brief events such as be critical for PDE10A responses in D2 MSNs, since we the phasic dopamine signal associated with reward and found no alteration of these responses in DARPP-32 T75A novelty (Schultz, 2010), while filtering out smaller fluctua- knock-in mutant mice. tions in basal cAMP level. The increased responsiveness of D2 MSNs at the level In contrast, in D2 MSNs DARPP-32 is phosphorylated of PKA signaling is opposed in vivo by the activity of D2 on the Thr34 residue, as previously demonstrated (Nishi receptors: the simple blockade of these receptors by D2 et al., 2008). In this situation, a moderate cAMP signal, antagonists strongly activates cAMP-dependent phos-

A B

Figure 6. The DARPP-32 T34 residue is required for a TP-10-induced increase of histone H3 phosphorylation in the striatum in adult mice in vivo. Wild type (WT) and DARPP-32 T34A mutant mice were treated with TP-10 (3 mg/kg) or vehicle. A, Examples of PH3 immunofluorescence, showing the dramatic reduction of TP-10 effects in the DARPP-32 T34A mutant mice. Scale bar, 20 ␮m. B, Quantification of the number of PH3-positive neurons in striatal coronal sections. Error bars indicate the SEM. Data were analyzed ϭ Ͻ ϭ Ͻ ϫ by a two-way ANOVA: genotype effect, F(1,12) 13.7, p 0.01; TP-10 effect, F(1,12) 16.1, p 0.01; genotype TP-10 interaction, Ͻ ؊3 ءءء ϭ Ͻ F(1,12) 14.8, p 0.01. Bonferroni’s post hoc test, p 10 .

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Figure 7. Diagram depicting the D1/D2 differential response to PDE10A inhibition. PDE10A inhibition increases cAMP and activates PKA to similar levels in D1 and D2 MSNs. In D2 MSNs, DARPP-32 is phosphorylated and inhibits PP-1: PKA substrates thus remain in the phosphorylated state, both in the cytosol and in the nucleus. In D1 MSNs, DARPP-32 is in a dephosphorylated state: PP-1 is fully active and dephosphorylates PKA substrates. Differences in PP2A/B activities between D1 and D2 MSNs may explain this imbalance.

phorylation (Håkansson et al., 2006; Bertran-Gonzalez striatal subregion is innervated by prefrontal and cingulate et al., 2008, 2009; Bonito-Oliva et al., 2011; Valjent et al., cortices, which are involved in the limbic system, and 2011). Accordingly, haloperidol or clozapine selectively dopamine neurons originating from the ventral tegmental increases phospho-Thr34-DARPP-32 in D2 MSNs, and area. D1 and D2 receptors exert contrasting roles selec- not in D1 MSNs (Bateup et al., 2008). This role of tively in the dorsomedial striatum during behavioral inhi- DARPP-32 in D2 MSNs is functionally important for some bition in the stop-signal task in rats (Eagle et al., 2011), effects of antipsychotic drugs since conditional knockout and lesions of the dorsomedial striatum disrupt prepulse of DARPP-32 in D2 MSNs leads to an increased locomo- inhibition (Baldan Ramsey et al., 2011). This region is also tor activity and a strongly reduced catalepsy upon admin- involved in early motor learning and is required for the istration of D2 receptor inhibitors (Bateup et al., 2010). Our cataleptic effects of haloperidol and for amphetamine data show that the inhibition of PDE10A has functional motor response sensitization (Durieux et al., 2012). Medial effects that are similar to the blockade of D2 receptors striatum thus appears as a limbic system-related region since both potentiate the PKA pathway selectively in D2 that could be affected in schizophrenia. MSNs. The particular sensitivity of D2 MSNs includes The differences observed between medial and lateral DARPP-32 Thr34 phosphorylation, which is more in- striatum likely involve network effects and differences in creased by PDE10A inhibitors in this population than in D1 synaptic plasticity reported between these two striatal MSNs (Nishi et al., 2008). Thus, hypersensitivity of subregions (Lovinger, 2010), and further work should pre- DARPP-32 Thr34 phosphorylation could be a critical fac- cisely show how these spatial differences are related to tor to account for the responsiveness of D2 MSNs to the behavior. Our work nonetheless shows that the D1/D2 blockade of either D2 receptors or PDE10A activity. difference is present in brain slices in both medial and The cellular effects of PDE10A inhibition affect MSNs lateral striatum, and remains when network activity is neuronal properties, which are integrated through the blocked, showing that the D1/D2 difference is an intrinsic basal ganglia network. For example, PDE10A inhibition property of D1 and D2 MSNs. The in vivo effects of was shown to potentiate D-amphetamine-dependent do- PDE10A inhibitors were totally abolished in T34- paminergic neuromodulation in vivo (Sotty et al., 2009). In DARPP-32 mutant mice, confirming the role of the addition, PDE10A inhibition was shown to massively in- DARPP-32/PP-1 loop as the initial determinant of the crease cGMP levels in vivo (Siuciak et al., 2006; Schmidt positive PKA response obtained during PDE10A inhibi- et al., 2008; Grauer et al., 2009), whereas, no effect was tion. observed in brain slice preparations (Nishi et al., 2008; this Further work is needed to determine whether the im- study). This discrepancy possibly results from the recruit- balance in PKA signal integration between D1 and D2 ment of striatal NOergic interneurons through network MSNs might be of interest to understand the pathophys- activity resulting indirectly from PDE10A inhibition. iology of other diseases that affect neuromodulatory pro- In vivo, the inhibition of PDE10A also selectively acti- cesses in basal ganglia such as Parkinson’s disease or vates D2 MSNs in the medial striatum. Interestingly, this Huntington’s disease (Threlfell and West, 2013).

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